FGF-18 compound dosing regimen

ABSTRACT

The present invention provides a new dosing regimen for administration of FGF-18 in the treatment of a cartilage disorder, such as osteoarthritis or cartilage injury. Specifically provided is a preferred treatment scheme comprising administrations every 3 weeks, 4 weeks or 5 weeks of an FGF-18 compound per treatment cycle. The new dosing regimen can include the co-adminsitration of an anti-inflammatory drug.

CROSS-REFERENCE TO RELATED APPLICATION

This application is the U.S. national stage application of InternationalPatent Application No. PCT/EP2015/053624, filed Feb. 20, 2015.

FIELD OF INVENTION

The present invention relates to the treatment of cartilage andcartilage/bone disorders, osteoarthritis and cartilage injury inparticular. More particularly, it relates to an FGF-18 compound for useaccording to particular treatment regimens. Specifically, it providestreatment schemes comprising administrations every 3 weeks, 4 weeks or 5weeks of an FGF-18 compound per treatment cycle. The dosing regimen canfurther comprise the administration of an anti-inflammatory drug witheffects on symptoms (pain and function), such as anakinra or diclofenac.

BACKGROUND OF THE INVENTION

Cartilage is composed of chondrocytes (cells derived from mesenchymalcells) which are dispersed in the matrix (a firm, gel-like groundsubstance). The cartilaginous matrix is produced by these cells andcomprises mainly Type II collagen fibers (except fibrocartilage whichalso contains Type I collagen fibers), proteoglycans, and elastinfibers. Cartilage is found, among other places, in the joints, theribcage, the ears, the nose, the throat, the trachea and theintervertebral disks. There are three main types of cartilage, hyaline,elastic and fibrocartilage, providing notably different functionalproperties according to their histological morphology. Articularcartilage, for instance, is a hyaline cartilage, having viscoelasticproperties, covering the articular surfaces of bones. The main purposeof articular cartilage is to provide smooth surfaces in order to ensurenearly frictionless movement of articulating bones.

Cartilage disorders broadly refers to diseases characterized bydegeneration/disintegration of cartilage and abnormalities in theconnective tissues which are manifested by inflammation, pain, stiffnessand limitation of motion of the affected body parts. These disorders canbe due to a pathology or can be the result of trauma or injury. Maturecartilage has very limited ability to self-repair, notably becausemature chondrocytes have little potential for proliferation due tolimited supply with nutrients linked to the absence of blood vessels incartilage. Replacement of damaged cartilage, in particular articularcartilage, caused either by injury or disease, is a major challenge forphysicians, and available surgical treatment procedures are consideredunpredictable and effective for only a limited time in younger patientswithout osteoarthritic changes. Therefore, the majority of patientseither do not seek treatment or are counseled to postpone treatment foras long as possible. When treatment is required, the standard procedureis age-dependent and varies between total or partial joint replacement,transplantation of pieces of cartilage or chondrocytes ormarrow-stimulating techniques (such as microfracture). Microfracture isa cheap and common procedure that involves penetration of thesubchondral bone to stimulate cartilage deposition by bonemarrow-derived stem cells. However, it has been shown that thistechnique does not sufficiently repair the chondral defect and the newcartilage formed is mainly fibrocartilage, resulting in a short-livedrepair tissue. Indeed, fibrocartilage does not have the samebiomechanical properties as hyaline articular cartilage and often lacksproper lateral integration into the surrounding cartilage. For thisreason, the newly synthesized fibrocartilage may break down more easily(expected time frame: 5-10 years).

For patients with osteoarthritis (OA) all these cartilage repairtechniques fail. The remaining non-surgical treatment consists notablyof physical therapy, lifestyle modification (e.g., body weightreduction), supportive devices, oral drugs (e.g., non-steroidalanti-inflammatory drugs), injection of drugs (e.g., hyaluronic acid andcorticoids), and food suplementation. All these treatments are unable tostop OA disease progression. If the pain therapy also fails, surgery,such as joint replacement or high tibial osteotomy for the knee joint,is the remaining option for the patients. Tibial or femoral osteotomies(cutting the bone to rebalance joint wear) may reduce symptoms, help tomaintain an active lifestyle, and delay the need for total jointreplacement. Total joint replacement can provide relief for the symptomsof advanced osteoarthritis, but generally requires a significant changein a patient's lifestyle and/or activity level.

Current available drug treatments are mainly directed to pain relief. Atthis time, there is no commercially available treatment that restoresthe cartilage damage (Lotz, 2010).

Interleukin-1 alpha (IL-1α) and interleukin-1 beta (IL-1β) are naturallyoccurring agonists of the type I IL-1 receptor (IL-1RA). Overexpressionof proinflammatory cytokines, such as IL-1, has been shown to play amajor role in the pathogenesis of immunoinflammatory diseases such asrheumatoid arthritis (RA) (Bingham, 2002) or osteoarthritis (OA) (Lee etal., 2013). The clinical application of antagonizing IL-1α and IL-1β inRA has been investigated with anakinra (Kineret™), a recombinant,non-glycoslyated form of human IL-1ra. The use of this therapeuticprotein has led to a reduction in frequency and severity of joint damagein RA patients (Bresnihan, 2002; St. Clair, 2002), as well as painreduction (Mertens et al., 2009). This molecule has been approved in2001 in the treatment of some types of RA. Although IL-1 is alsoinvolved in OA, anakinra therapy is not significantly associated withimprovements in OA symptoms compared with placebo, although a tendencytoward pain reduction with anakinra 150 mg versus placebo was noted(Chevalier et al., 2009).

Fibroblast Growth Factor 18 (FGF-18) is a member of the FibroblastGrowth Factor (FGF) family of proteins, closely related to FGF-8 andFGF-17. It has been shown that FGF-18 is a proliferative agent forchondrocytes and osteoblasts (Ellsworth et al., 2002; Shimoaka et al.,2002). FGF-18 has been proposed for the treatment of cartilage disorderssuch as osteoarthritis and cartilage injury, either alone(WO2008/023063) or in combination with hyaluronic acid (WO2004/032849).

Various dosing regimens have been suggested for FGF-18. For instance,Moore et al. (2005) disclosed administration twice weekly for 3 weeks,and WO2008/023063 taught administration once a week for 3 weeks. Thislast dosing regimen has been investigated in clinical trials (for moredetails see for instance NCT01033994, NCT00911469 and NCT01066871).

Although the dosing regimen described in WO2008/023063 gives goodresults in articular cartilage repair, there is still a risk of acutesynovitis. For this reason, there is a need for a method of decreasingrisk of treatment-related acute synovitis as well as increasing patienttolerance to intra-articular injection, while maintaining the efficacyfor the treatment of cartilage disorders, notably via chondrocyteproliferation and subsequent cartilage repair. Such a method should notonly allow articular cartilage repair, possibly in the absence ofsynovitis, but also allow reformation of new cartilage having goodproperties (i.e., mainly hyaline cartilage). Indeed, generation of saidhyaline cartilage is valuable both as a therapeutic and as a componentfor biological matrices (Getgood et al., 2010). There is also a need fora method of decreasing pain/improving function, while maintaining theefficacy for the treatment of cartilage disorders. Indeed, pain is notonly very often associated with cartilage disorders but represents theleading symptom for clinical detection of these disorders.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide a method fortreating a patient having a cartilage disorder comprising theadministration of an FGF-18 compound, wherein the FGF-18 compound isadministered at least two times per treatment cycle, saidadministrations being separated by about 3 to 5 weeks, preferably by amonth (monthly administrations). In a preferred embodiment saidadministrations are separated by regular interval of about 3, 4 or 5weeks each. Preferably, the FGF-18 compound is administered at regularintervals of once per month.

The present invention further provides an FGF-18 compound for use in thetreatment of a patient having a cartilage disorder, wherein the FGF-18compound is to be administered at least two times per treatment cycle,said administrations being separated by about 3 to 5 weeks, preferablyby about one month (monthly administrations). In a preferred embodimentsaid administrations are separated by regular interval of about 3, 4 or5 weeks each. Preferably, the FGF-18 compound is administered at regularintervals of once per month

Preferably, the FGF-18 compound to be administered is the FGF-18fragment designated herein as trFGF-18 (or sprifermin) and the posologycycle is 3 to 300 μg per intra-articular injection, once monthly for 3consecutive months (one treatment cycle).

In the context on the present invention as a whole, any treatment (orany method for treating) with an FGF-18 compound can further comprisethe administration of an anti-inflammatory drug with effects on symptoms(pain and function), such as anakinra or diclofenac. Preferably, suchadministration is performed at the same time as the administration ofthe FGF-18 compound. If an anti-inflammatory drug is needed, it wouldtherefore follow that said anti-inflammatory drug is to be administeredconcomitantly with the FGF-18 compound at least two times per treatmentcycle, said administrations being separated by about 3 to 5 weeks,preferably by about one month (monthly administrations). Preferably theanti-inflammatory drug is anakinra or diclofenac and the posology cycleis 0.01 to 500 mg per administration, once monthly for 3 consecutivemonths (one treatment cycle).

In a preferred embodiment such a treatment cycle (i.e., FGF-18 compoundalone or in combination with an anti-inflammatory drug with effects onsymptoms (pain and function)) may be repeated several months after thelast injection of the preceding treatment cycle, for instance after 2, 4or 6 months or even after 1 year.

In preferred embodiments of the invention, the cartilage disorder to betreated is arthritis, such as osteoarthritis, or cartilage injury withor without surgical intervention such as microfracture. It hassurprisingly been found that the methods and uses of the presentinvention notably improve cartilage repair and stimulate proliferationof chondrocytes, and, in the meantime, increase patient tolerance tointra-articular injection. It has further surprisingly been found thatwhen anakinra or diclofenac is used together with FGF-18, it is able toefficiently restore the proliferative activity of the FGF-18 compound.Another advantage of the present invention is that synovitis will bereduced compared to what is observed with other regimens.

DEFINITIONS

The term “FGF-18 compound” or “FGF-18”, as used herein, is intended tobe a protein maintaining at least one biological activity of the humanFGF-18 protein. FGF-18 may be native, in its mature form, a recombinantform or a truncated form thereof. Biological activities of the humanFGF-18 protein notably include the increase in chondrocyte or osteoblastproliferation (see WO98/16644) or cartilage formation (seeWO2008/023063). Native, or wild-type, human FGF-18 is a proteinexpressed by chondrocytes of articular cartilage. Human FGF-18 was firstdesignated zFGF-5 and is fully described in WO98/16644. SEQ ID NO: 1corresponds to the amino acid sequence of the native human FGF-18, witha signal peptide consisting of amino acid residues 1(Met) to 27(Ala).The mature form of human FGF-18 corresponds to the amino acid sequencefrom residue 28(Glu) to residue 207(Ala) of SEQ ID NO: 1 (180 aminoacids).

FGF-18, in the present invention, may be produced by a recombinantmethod, such as taught by WO2006/063362. Depending on the expressionsystems and conditions, FGF-18 in the present invention is expressed ina recombinant host cell with a starting Methionine (Met) residue or witha signal sequence for secretion. When expressed in a prokaryotic host,such as E. coli, FGF-18 contains an additional Met residue in theN-terminal of its sequence. For instance, the amino acid sequence ofhuman FGF-18, when expressed in E. coli, starts with a Met residue inN-term (position 1) followed by residues 28(Glu) to residue 207(Ala) ofSEQ ID NO: 1.

The term “truncated form of FGF-18”, as used herein, refers to a proteinwhich comprises or consists of residues 28(Glu) to 196(Lys) of SEQ IDNO: 1. Preferably, the truncated form of FGF-18 protein is thepolypeptide designated “trFGF-18” (170 amino acids; also known asrhFGF-18 or sprifermin), which starts with a Met residue (in theN-terminal) followed by amino acid residues 28(Glu)-196(Lys) of thewild-type human FGF-18. The amino acid sequence of trFGF-18 is shown inSEQ ID NO: 2 (amino acid residues 2 to 170 of SEQ ID NO: 2 correspond toamino acid residues 28 to 196 of SEQ ID NO: 1). trFGF-18 is arecombinant truncated form of human FGF-18, produced in E. coli (seeWO2006/063362). trFGF-18 has been shown to display similar activities tothe mature human FGF-18, e.g., it increases chondrocyte proliferationand cartilage deposition, leading to repair and reconstruction for avariety of cartilaginous tissues (see WO2008/023063).

The terms “anti-inflammatory drug with effects on symptoms (pain andfunction)” or “anti-inflammatory drug” as used herein refer to ananti-inflammatory drug having effects on symptoms linked to thecartilage disorders to be treated such as pain and function. Thepreferred anti-inflammatory drugs to be used according to this inventionare anakinra and diclofenac. Anakinra is a recombinant, nonglycosylatedform of human interleukin-1 receptor antagonist (IL-1Ra). It iscommercialized under the name Kineret®. Its sequence corresponds to SEQID NO: 3. Diclofenac (i.e., 2-(2,6-dichloranilino) phenylacetic acid) isa drug well-known for reducing inflammation and as an analgesic reducingpain in certain conditions. It is commercialized under various tradenames.

The term “about” in “about 3 to 5 weeks” or “about one month”encompasses administrations separated by 3 weeks (21 days) to 5 weeks(35 days) or by one month, as well as administrations separated by 3 to5 weeks+/−a few days or by one month+/− a few days (e.g., +/−1, 2, 3, 4day(s)). Indeed, it should be understood that, notably from a practicalpoint of view, the administration of the FGF-18 compound, for instancetrFGF-18, cannot always be performed at exact intervals, e.g., exactly 4weeks (28 days) day per day after the previous administration.Therefore, in the context of the invention, 4 weeks means 28 days, butmay also be 24, 25, 26, 27, 28, 29, 30, 31 or 32 days after the previousadministration, for the convenience of the patient. In the context ofthe present invention, the term “4 weeks” is similar to the term “1month” and they can be used interchangeably (FIG. 1). “4 weeks” will bepreferably used should one refer to “days” (e.g. first injection on aMonday, following injection on a Monday 4 weeks after) and “month” willbe preferably used should one refer to a “date” (e.g., first injectionthe 1^(st) of August, following injection the 1^(st) of September).

The term “treatment cycle” or “cycle” corresponds to the period whereinan FGF-compound is given every 3, 4 or 5 weeks or every month(consecutive administrations). As an example, a treatment cycle canconsist of 3 injections at 3-week intervals or can consist of 3injections at 4-week intervals. Such a “treatment cycle” can berepeated. For instance, a second “treatment cycle” can be performed 3,4, 5 or 6 months after the last injection of the previous cycle.Alternatively, a second cycle can also be performed 1 year or 2 yearsafter the first injection in the first cycle. As an example, a firsttreatment cycle consisting of 3 injections at 4-week intervals can befollowed, 3 months after the last injection of said cycle, by a secondtreatment cycle of 3 injections at 4-week intervals.

The term “cartilage disorder”, as used herein, encompasses disordersresulting from damage due to injury, such as traumatic injury,chondropathy or arthritis. Examples of cartilage disorders that may betreated by the administration of the FGF-18 formulation described hereininclude but are not restricted to arthritis, such as osteoarthritis, andcartilage injury. Degenerative diseases/disorders of the cartilage orjoint, such as chondrocalcinosis, polychondritis, relapsingpolychondritis, ankylosing spondylitis or costochondritis, are alsoencompassed by this wording. The International Cartilage Repair Societyhas proposed an arthroscopic grading system to assess the severity ofthe cartilage defect: grade 0: (normal) healthy cartilage, grade 1: thecartilage has a soft spot or blisters, grade 2: minor tears visible inthe cartilage, grade 3: lesions have deep crevices (more than 50% ofcartilage layer) and grade 4: the cartilage tear exposes the underlying(subchronal) bone (see ICRS publication: see Worldwide Website:cartilage.org/_files/contentmanagement/ICRS_evaluation.pdf, page 13).

-   -   The term “arthritis” as used herein encompasses disorders such        as osteoarthritis, rheumatoid arthritis, juvenile rheumatoid        arthritis, infectious arthritis, psoriatic arthritis, Still's        disease (onset of juvenile rheumatoid arthritis) or        osteochondritis dissecans. It preferably includes diseases or        disorders in which the cartilage is damaged.

The term “osteoarthritis”, or “OA”, is used to intend the most commonform of arthritis. The term “osteoarthritis” encompasses both primaryosteoarthritis and secondary osteoarthritis (see for instance The MerckManual, 17^(th) edition, page 449). Osteoarthritis may be caused by thebreakdown of cartilage. Bits of cartilage may break off and cause painand swelling in the joint between bones. Over time, the cartilage maywear away entirely, and the bones will rub together. Osteoarthritis canaffect any joint but usually concerns hands, shoulders andweight-bearing joints such as hips, knees, feet, and spine. In apreferred example, the osteoarthritis may be knee osteoarthritis or hiposteoarthritis. This wording notably encompasses the forms ofosteoarthritis which are classified as stage 1 to stage 4 or grade 1 tograde 6 according to the OARSI classification system. The skilled personis fully aware of osteoarthritis classifications that are used in theart, in particular said OARSI assessment system (also named OOCHAS; seefor instance Custers et al., 2007). Osteoarthritis is one of thepreferred cartilage disorders that can be treated by administering theFGF-18 compounds according to the present invention.

The term “cartilage injury” as used herein is a cartilage disorder orcartilage damage resulting, notably, from trauma. Cartilage injuries canoccur notably after traumatic mechanical destruction, notably further toan accident or surgery (for instance microfracture surgery). The term“cartilage injury” also includes chondral or osteochondral fracture anddamage to the meniscus. Also considered within this definition issport-related injury or sport-related wear of tissues of the joint. Theterm also includes microdamage or blunt trauma, a chondral fracture, anosteochondral fracture or damage to the meniscus.

In the context of the present invention, the “efficacy” of a treatmentcan be measured based on changes in the thickness of the cartilage, forinstance the thickness of the articular cartilage of the joint. Thisthickness can be assessed, for instance, through X-ray computedtomography, Magnetic Resonance Imaging (MRI) or ultrasonic measurements.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides administration schemes for the treatmentof various cartilage disorders, such as osteoarthritis and cartilageinjury, with an FGF-18 compound. Preferably, said FGF-18 compound istrFGF-18 as defined above. In the context of the present invention ithas been shown that FGF-18 compounds have optimal disease- orsymptom-ameliorating effects on cartilage disorders when administeredaccording to the methods and uses disclosed herein. It has beensurprisingly found that the dosing regimens of the present invention(i.e., less frequent dosing schedules than once a week for three weeksper treatment cycle) cause reduced inflammation (e.g., acute synovitis)status during the next injection and therefore the full activity ofFGF-18 compounds can be observed. This finding was not expected becauseof the rather short half-life of FGF-18 in the joint (less than 24hours).

In the context of the present invention as a whole, any treatment (orany method for treating) with an FGF-18 compound can further comprisethe administration of an anti-inflammatory drug with effects on symptoms(pain and function), such as anakinra or diclofenac. Preferably, suchadministration (or co-administration) is performed at the same time as(i.e., concomitantly with) the administration of the FGF-18 compound.

In one embodiment, the present invention provides a method for treatinga patient having a cartilage disorder comprising the administration ofan FGF-18 compound wherein the FGF-18 compound is administered at leasttwo times per treatment cycle, said administrations being separated byabout 3 to 5 weeks, preferably by about 1 month. The preferred FGF-18compound is trFGF-18. In a preferred embodiment the FGF-18 compound isadministered for 3 consecutive months per treatment cycle (i.e., 3 timesper treatment cycle, at regular intervals of once per month or onceevery four weeks). Alternatively, the FGF-18 compound can beadministered 3 times per treatment cycle, at regular intervals of 3weeks or 5 weeks. Such treatment may comprise 1, 2 or 3 treatment cyclesper year. An anti-inflammatory drug, such as anakinra or diclofenac, canbe administered at the same time as the FGF-18 compound.

In another aspect of the present invention there is provided an FGF-18compound for use in the treatment of a patient having a cartilagedisorder, wherein the FGF-18 compound is administered at least two timesper treatment cycle, said administrations being separated by about 3 to5 weeks, preferably by about 1 month. The preferred FGF-18 compound istrFGF-18. In a preferred embodiment the FGF-18 compound is administeredfor 3 consecutive months per treatment cycle (i.e., 3 times pertreatment cycle, at regular intervals of once per month or once everyfour weeks). Alternatively, the FGF-18 compound can be administered 3times per treatment cycle, at regular intervals of 3 weeks or 5 weeks.Such treatment may comprise 1, 2 or 3 treatment cycles per year. Ananti-inflammatory drug with effects on symptoms (pain and function),such as anakinra or diclofenac, can be administered at the same time asthe FGF-18 compound.

According to the present invention, the administration of the FGF-18compound, either alone or in combination with an anti-inflammatory drug,such as anakinra or diclofenac, is to be performed at regular intervals;however, slight variations of +/− a few days are authorized (preferablyno more than 3 or 4 days). For example, where administrations areseparated by about 4 weeks, if the first administration of a cycle isgiven on a Tuesday, the second administration may be made on the Tuesday4 weeks after the first administration (regular interval) or a few daysbefore or after (for instance the Monday before or Thursday after).Similarly, where administrations are separated by about 1 month, if thefirst administration is given for instance on the 1^(st) of August, thesecond administration may be made on the 1^(st) of September (aThursday, for instance), i.e., one month after the first administration(regular interval) or a few days before or after (for instance theMonday, Tuesday or Wednesday before the 1^(st) of September or theFriday or Monday following he 1^(st) of September). Such flexibilityallows the dosing regimen to be notably less restricting and moreconvenient for the patient.

Preferably, administrations are performed on a regular interval basis,e.g., every 3, 4 or 5 weeks or every month. In a particular embodiment,said administrations are separated by about 1 month (or about 4 weeks).In one preferred embodiment they are separated by 1 month or by 4 weeks(i.e., monthly injections). Preferably, the FGF-18 compound is to beadministered at regular intervals once per month (or once every 4weeks). Alternatively, the FGF-18 compound can be administered atregular intervals once every 3 weeks or once every 5 weeks.

In the context of the present invention as a whole, the FGF-18 compoundis administered at least two times per treatment cycle. It can also beadministered, for instance, at least 3 times or at least 4 times pertreatment cycle. Preferably, it is administered 3 times or 4 times pertreatment cycle.

In a preferred embodiment, the FGF-18 compound, either alone or incombination with an anti-inflammatory drug (such as anakinra ordiclofenac), is to be administered for at least 2 consecutive months, atleast 3 consecutive months or at least 4 consecutive months pertreatment cycle. In a further preferred embodiment the FGF-18 compound,either alone or in combination with an anti-inflammatory drug (such asanakinra or diclofenac), is administered for 2 consecutive months, 3consecutive months or 4 consecutive months per treatment cycle. In aneven preferred embodiment, it is administered for 3 consecutive months.

In the context of the present invention as a whole, such treatment maycomprise several treatment cycles per year, such as 1, 2 or 3 treatmentcycles per year. In one preferred embodiment, such treatment comprises 2cycles per year. As an alternative, the treatment comprises 1 cycle peryear, repeated 1 year or 2 years after the beginning of the firsttreatment cycle. As an example, should a treatment comprise 1 cycle,said treatment may consist of 3 injections at 3-week intervals or mayconsist of 3 injections at 4-week intervals. As a further example,should a treatment comprise at least 2 cycles, a first treatment cycleconsisting of 3 injections at 3-week intervals can be followed, severalmonths after the last injection of said cycle, by a second treatmentcycle of 3 injections at 3-week intervals; or a first treatment cycleconsisting of 3 injections at 4-week intervals can be followed, severalmonths after the last injection of said cycle, by a second treatmentcycle of 3 injections at 4-week intervals.

The FGF-18 compound of the invention is preferably selected from thegroup consisting of a) a polypeptide comprising or consisting of thehuman FGF-18 mature form comprising residues 28-207 of SEQ ID NO: 1, orb) a polypeptide comprising or consisting of FGF-18 (170AA) (SEQ ID NO:2). Particularly, this compound is selected from human wild-type matureFGF-18 or trFGF-18. Said compound increases cartilage deposition andallows cartilage repair.

In a further preferred embodiment, the treatment comprisesadministration of the FGF-18 compound at a dose of 3-600 micrograms (μgor mcg), or preferably 3-300 μg, or preferably 10-200 μg, or morepreferably 30-150 μg, or even more preferably 30-120 μg per singleintra-articular administration. In a preferred embodiment the treatmentcomprises administration at a dose of about 3, 10, 20, 30, 40, 50, 60,90, 100, 120, 150, 180, 200, 240 or 300 μg per single intra-articularadministration of the FGF-18 compound. Preferred doses include 10, 20,30, 60, 90, 120, 180, 240 or 300 μg per single intra-articularadministration of the FGF-18 compound. It should be understood that thedose of the FGF-18 compound to be administered will be different if thepatient to be treated is a human or a non-human mammal. For instance,for dogs, the dose will be preferably 5-fold less important than forhumans. As an example, should the human dose range from 30 to 120 μg persingle intra-articular administration, the dose for a dog could rangefrom 5 to 20 μg per single intra-articular administration. Examples ofdosing for rats and rabbits can be found in the Examples section.

In the context of the present invention as a whole, the FGF-18 compoundcan be used in combination with an anti-inflammatory drug with effectson symptoms (pain and function). The preferred anti-inflammatory drug isanakinra (see SEQ ID NO: 3) or diclofenac. When any one of anakinra ordiclofenac is administered, the treatment comprises administration at adose of 0.01-500 milligrams (mg), preferably 0.1-250 mg, or morepreferably 0.5-150 mg per single administration, eitherintra-articularly (the preferable way for anakinra, for instance) ororally (the preferable way for diclofenac, for instance). In a preferredembodiment the treatment comprises administration at a dose of about0.03, 0.1, 0.25, 0.3, 0.5, 1, 1.5, 2, 5, 10, 15, 20, 30, 40, 50, 60, 70,80, 90, 100, 150, 200, 250 or 300 mg per single administration of theanti-inflammatory drug. Preferred doses include 0.5, 1, 5, 1.5, 10, 50,100 and 150 mg per single administration. It should be understood thatthe dose of anti-inflammatory drug to be administered will be differentif the patient to be treated is a human or a non-human mammal. Forinstance, for dogs, the dose will be preferably 6-fold less than forhumans. As an example, if the human dose of anakinra is 150 mg persingle intra-articular administration, the dose for a dog could be 25 mgper single intra-articular administration. The physician will adapt thedosing regimen for the anti-inflammatory drug case by case, depending onthe patient and the anti-inflammatory drug to be administered. Forinstance, for diclofenac given orally, the dosing regimen can be 50 or75 mg, two or three times a day.

FGF-18 compounds may be formulated as a pharmaceutical composition,i.e., together with a pharmaceutically acceptable carrier, excipient orthe like. The definition of “pharmaceutically acceptable” is meant toencompass any carrier, excipient or the like which does not interferewith the effectiveness of the biological activity of the activeingredient and that is not toxic to the patient to which it isadministered. For example, for parenteral administration, the activeprotein(s) may be formulated in a unit dosage form for injection invehicles such as saline, dextrose solution, serum albumin and Ringer'ssolution. Formulations for intra-articular application will comply withmost of the requirements that also apply to other injectionformulations, i.e., they need to be sterile and compatible with thephysiological conditions at the application site (e.g., knee joint,synovial fluid). The excipients used for intra-articular injection mayalso be present in other injection formulations, e.g., for intramuscularor subcutaneous application. Such formulations of FGF-18 compounds,including at least one further pharmaceutically acceptable carrier,excipient or the like, are herein also referred to as “FGF-18compositions” or “FGF-18 formulations”. Said “FGF-18 compositions” or“FGF-18 formulations” are also useful in the context of the presentinvention.

If an anti-inflammatory drug, such as anakinra or diclofenac, is usedtogether with an FGF-18 compound, it can either be added to the FGF-18compound formulation before administration, or co-administered, eitherusing 2 different syringes/needles, or using 2 different syringes butpreferably the same needle to increase patient convenience.Alternatively, the anti-inflammatory drug can be administered orally orby any other way of administering.

FGF-18 compounds, such as trFGF-18, and compositions containing FGF-18compounds (“FGF-18 compositions”) will be useful for treating cartilagedisorders. In particular it can be useful for treating articularcartilage defects in synovial joints that are, for instance, due tosuperficial fibrillation (early osteoarthritis), cartilage degenerationdue to osteoarthritis, and chondral or osteochondral defects due toinjury or disease. FGF-18 compounds and compositions may also be usefulfor treating joint disease caused by osteochondritis dissecans anddegenerative joint diseases. In the field of reconstructive and plasticsurgery, FGF-18 compounds and compositions will be useful for autogenousor allogenic cartilage expansion and transfer for reconstruction ofextensive tissue defects. FGF-18 compositions can be used to repaircartilage damage in conjunction with lavage of the joint, stimulation ofbone marrow, abrasion arthroplasty, subchondral drilling, ormicrofracture of the subchondral bone. The optional co-administrationwith an anti-inflammatory drug with effects on symptoms (pain andfunction), such as anakinra or diclofenac, would decrease painassociated with the cartilage disorder to be treated.

In a preferred embodiment, the cartilage disorder to be treatedaccording to the invention is osteoarthritis, such as kneeosteoarthritis or hip osteoarthritis. The osteoarthritis to be treatedcan be, for example, and not limited to, primary osteoarthritis orsecondary osteoarthritis, as well as osteoarthritis which is classifiedas stage 1 to stage 4 or grade 1 to grade 6 according to the OARSIclassification system.

In another preferred embodiment, the cartilage disorder to be treatedaccording to the invention is cartilage injury with and without surgicalinterventions, such as microfractures. Additionally, after the growth ofcartilage due to the administration of the FGF-18 composition(administered either alone or together with an anti-inflammatory drug,such as anakinra or diclofenac), a surgical treatment may be necessaryto suitably contour the newly formed cartilage surface.

In a preferred embodiment, the treatment comprises intra-articularadministration of the FGF-18 compound or FGF-18 composition, eitheralone or together with an anti-inflammatory drug with effects onsymptoms (pain and function), such as anakinra or diclofenac. FGF-18compounds or FGF-18 compositions can be applied, either alone ortogether with an anti-inflammatory drug, such as anakinra or diclofenac,by direct injection into the synovial fluid of the joint or directlyinto the defect, either alone or complexed with a suitable carrier forextended release of protein (e.g., sustained-release formulations) orrestricted local release. Preferably, the mode of administration of theFGF-18 compound, either alone or together with an anti-inflammatorydrug, such as anakinra or diclofenac, described herein is selected fromthe group consisting of peri-synovial administration, intra-synovialadministration, peri-articular administration and intra-articularadministration. In a preferred embodiment, the FGF-18 compound describedherein is administered, either alone or together with ananti-inflammatory drug, such as anakinra or diclofenac, preferablyintra-articularly (administration within a joint). The anti-inflammatorydrug is administered preferably intra-articularly (the preferable wayfor anakinra) or orally (the preferable way for diclofenac). Theintra-articular administration is done in a joint selected from thejoint of the hip, knee, elbow, wrist, ankle, spine, foot, finger, toe,hand, shoulder, rib, shoulder blade, thigh, shin, heel and along thebony points of the spine. In yet another preferred embodiment theintra-articular administration is done in a joint of the hip or theknee.

DESCRIPTION OF THE FIGURES

FIGS. 1A-1C: Treatment schedule outline. FIG. 1A: once weekly dosingregimen; FIG. 1B: once monthly (or every 4 weeks) dosing regimen; FIG.1C: once monthly (or every 4 weeks) dosing regimen+/−a few days ofvariation accepted for the convenience of the patient.

FIGS. 2A-2B: FGF-18 compounds evoke dose-dependent articular matrixdeposition in a rat OA model illustrated by quantification of Safranin-Ostaining.

FIG. 3: Total Joint Score Without Femur.

FIG. 4: Total Joint Score.

FIG. 5: Substantial Tibial Cartilage Degeneration Width.

FIGS. 6A-6B: Cell concentration after 8 days of culture of porcinechondrocytes stimulated or not with IL1α and stimulated or not with IL1days of culture (A) or after a 6-hour delay (B) with rhFGF-18 100 ng/mLand/or anakinra 100 ng/mL. N=4 for (A) and N=6 for (B).

FIG. 7: Serum α2MG Levels Over Time.

FIG. 8: AUC (area under the curve) of joint diameter difference betweentarget knee (OA joint) and the contralateral joint (healthy knee) overtime.

FIG. 9: cartilage volume (in mm³) on medial tibia.

DESCRIPTION OF THE SEQUENCES

SEQ ID NO: 1: Amino acid sequence of the native human FGF-18.

SEQ ID NO: 2: Amino acid sequence of the recombinant truncated FGF-18(trFGF-18).

SEQ ID NO: 3: Amino acid sequence of the recombinant human ilL-1receptor antagonist (anakinra).

EXAMPLES

Material

The recombinant truncated FGF-18 (trFGF-18) of the present examples hasbeen prepared by expression in E. coli, according to the techniquedescribed in WO2006/063362. In the following examples, trFGF-18 andFGF-18 are used interchangeably.

The recombinant human interleukin-1 receptor antagonist (anakinra) hasbeen obtained via Pharmacia.

Scoring of Joints

After the sacrifice, the right knee was removed and decalcified in 5%formic acid for 4-6 d before cutting in half in the frontal plane andembedding in paraffin wax. Three sections were cut in 200 μm steps,stained with toluidine blue, and analyzed using ImagePro Plus™ software(Media Cybernetics). In scoring the three sections of the joint, theworst-case scenario for the two halves on each slide was determined forthe cartilage lesion as the cartilage degeneration width (μm). Thisreflects the areas of tibial cartilage lesion in which both chondrocyteand proteoglycan loss extend 50% of the cartilage thickness. Themeasurement was taken over the area of greatest lesion severity in eachof the three zones across the tibial surface.

A treatment group Mean±SE for each score and measurement was determined.

Data was analyzed using a one-way analysis of variance (1-way ANOVA) orKruskal-Wallis test (non-parametric), along with an appropriate multiplecomparison post-test. Right knee caliper measurements were compared toleft using a Student's t-test. Unless indicated, Bolder BioPATH, Inc.performs statistical analysis on raw (untransformed) data only.Statistical tests make certain assumptions regarding the data'snormality and homogeneity of variance, and further analysis may berequired if testing resulted in violations of these assumptions.Significance for all tests was set at p≦0.05. Comparisons were madebetween each group and the vehicle control group, as well as betweenpairs

Example 1

Method:

The anterior cruciate ligament transection with resection of the medialmeniscus (ACLT+tMx) model of instability-induced OA was performed inmale Lewis rats 10-15 weeks of age. Briefly, under anesthesia withIsoflurane, the joint capsule of the right knee of each rat was opened,the anterior cruciate and the meniscus fixating ligaments were sharplytransected, the meniscus was removed and the capsule, muscles and skinclosed by sutures. Animals were randomly allocated into 10 groups ofn=10 each. The following dosages were investigated: 0, 0.3, 1, 3 and 10μg per intra-articular injection. Groups 1-5 received one cycle of threesingle injections at weekly intervals, group 6-10 three singleinjections at monthly intervals. Intra-articular treatment was startedthree weeks post-surgery. At this time pathophysiological changes arealready mainifest, e.g., cartilage matrix loss. Animals were euthanized17 weeks after surgery and joints were investigated.

Results:

trFGF-18 induced dose-dependent neo-formation of cartilaginous tissuewhich became significant in the lateral tibia with 1 μg/jointindependently of the injection regime (see FIG. 2a ). trFGF-18 induceddose-dependent neo-formation of cartilaginous tissue which becamesignificant in the medial tibia with 1 μg/joint according to theone-weekly regimen and with 0.3 μg/joint according to the one-monthlyregimen (see FIG. 2b )

Example 2

Method:

Male Lewis rats underwent surgery (under anesthesia with Isoflurane) toinduce a medial meniscal tear in the right knee joint. Animals weredosed IA with vehicle or FGF-18 on one of two different regimens (seetable below), then terminated on day 105. Knees had caliper measurementstaken at baseline and days 21, 42, 56, 84, and 105. Serum was evaluatedfor α2 macroglobulin levels at baseline and on days 21, and 105, as wellas one week after the final dose (day 42 or 84). Right knees werecollected for histopathology evaluation.

TABLE 1 treatment groups Number Group of rats Treatment (10 μg in 50 μl)1 10 Normal + vehicle d 21, 35, 49 2 10 surgery + vehicle d 21, 35, 49 310 Surgery + FGF-18 d 21, 28, 35 (weekly treatment) 4 10 surgery +FGF-18 d 21, 35, 49 (bi-weekly treatment) 5 10 Surgery + FGF-18 d 21,49, 77 (monthly treatment) 6 10 Surgery + FGF18 d 21, 35 7 10 Surgery +FGF18 d 21, 77Results and Conclusions:

All animals resumed weight bearing immediately post-surgery uponrecovery from anesthesia. All animals gained weight over the course ofthe study and there were no significant differences in body weightchange between groups.

Knee diameters, measured by caliper measurement and serum levels ofa2MG, increased after FGF-18 injections but a clear benefit of onespecific treatment regimen over another was not demonstrated in thisstudy using these parameters. In contrast the structural benefit washighest with a monthly injection scheme (FIGS. 3, 4, 5).

Overall, results of this study indicate that treatment with FGF-18,regardless of regimen, significantly increased swelling in the treatedknee after the first injection. Histopathology evaluation indicated thattreatment generally increased the extent of injury over the tibialsurface, particularly in terms of collagen damage, while reducing itsseverity. This effect was most evident in the treatment groups thatreceived three doses rather than two, and in groups that received theirfinal dose at the latest time point (day 77). Several other side effectsof treatment were seen, including cartilage hypertrophy, synovitis, andsynovial fibrosis. These changes were most evident and severe in animalsthat received three doses in rapid succession (Groups 3 and 4), whileanimals that received only two doses (Groups 6 and 7) or received theirdoses over a greater timeframe (Group 5) had slightly less severechanges.

The treatment regimen that resulted in the best overall morphology wasthe three injections, once monthly (Group 5; injections at days 21, 49,and 77), indicating that some recovery time between doses wasbeneficial.

Example 3

Methods:

60 adult male, naïve, New Zealand White rabbits were used for thisstudy. Animals were approximately 3-4 months of age. Rabbits underwentsurgery for creation of the cartilage defect right knee. A 2 mm by 6 mmfull-thickness defect was made in the articular cartilage of thetrochlear groove. The microfracture was created using 18 gauge needles.Two 3 mm deep microfractures were made through the subchondral bone atthe base of the defect, one proximally and one distally. Eachmicrofracture hole was approximately 1.5 mm in diameter and the holesseparated by 2 mm.

The experiment consisted of the following six treatment groups, with 10animals in each group for a total of 60 rabbits (Table 2). Rabbits inGroups 3, 4, 5 and 6 were treated with intra-articular rhFGF-18 ineither one cycle of three weekly injections of 100 μg of rhFGF-18 (Group3 and 5) or one cycle of three monthly injections of 100 μg of rhFGF-18(Group 4 and 6). All animals were euthanized and necropsied 6 monthsafter surgery.

TABLE 2 treatment groups Dose Test article & μg/injection trFGF-18 Group# Cartilage Micro- treatment (trFGF- concentration Volume Day of (n =10) defect fracture schedule 18) (μg/ml) (ml/joint) dose 1 Yes No None 0NA NA NA 2 Yes Yes None 0 NA NA NA 3 Yes No rhFGF18 100 500 0.2 Day(1×/week for 3 7, 14, 21 weeks 4 Yes No 1×/month for 3 100 500 0.2 Daymonths) 28, 56, 84 5 Yes Yes rhFGF18 100 500 0.2 Day (1×/week for 3 7,14, 21 weeks 6 Yes Yes 1×/month for 3 100 500 0.2 Day months) 28, 56, 84Results:

Necropsy Findings (Table 3):

The ICRS gross cartilage score was 1.9+/−0.3 SE for Group 5 animals and2.4+/−0.2 SE for Group 6 animals, indicating that the monthly injectionscheme was superior compared to the weekly one. In Group 6 animals theintercondylar groove was filled in 3/10 animals with finely granular togranular cartilage; the remaining 7/10 animals had the groove 50% filledto nearly completely filled with granular to coarsely granularcartilage. In summary, the injection of rhFGF-18 alone or in combinationwith microfracture, administered as a single weekly or monthlyinjection, resulted in thickening or enlargement of the femoral condyleregion and a proliferation of rough or coarsely granular cartilage inthe intercondylar groove lesion and stimulation of osteophytes on themedial and lateral trochlear ridges, occasional tibial plateauosteophyte formation, or abnormal cartilage growth on the patella oradjacent to the patella in the synovial fat pad.

With No Microfracture:

Right femur lesion sections from animals treated monthly for threemonths with rhFGF-18 had significantly increased defect fill percentages(48%), resulting in a significant 49% increase in the defect fill score.Other scores were non-significantly increased by 20-23%. The total widthof the groove lesion was non-significantly increased by 36%, while thewidth of the lesion with no viable cartilage was significantly decreasedby 94%. All sections had degeneration that ranged from minimal tosevere, was generally focal, and was typically seen in conjunction withfibrocartilage. The central MFC thickness was significantly increased by120%.

Morphological Pathology with Microfracture:

Sections of the cartilage lesion area from untreated controls withmicrofracture had overall moderate reconstitution of the osteochondraljunction, moderately reduced matrix staining, and 50% fill of thedefect. Cell morphology was mostly fibrocartilage.

Lesion area sections from animals treated weekly for three weeks withrhFGF-18 had significant 44-69% increases (toward normal) in all scoredparameters, as well as the defect fill percent. Summed scores weresignificantly increased by 53%. The total width of the groove lesion wasslightly increased by 5%, while the width of the lesion with no viablecartilage was decreased (non-significantly) by 59%. All sections hadcartilage hypertrophy. Central MFC thickness in these sections wassignificantly increased by 112%.

Lesion area sections from animals treated monthly for three months withrhFGF-18 had significant 61-97% increased (toward normal) in all scoredparameters, as well as the defect fill percent. Summed scores weresignificantly increased by 76%. The total width of the groove lesion andthe width of the lesion with no viable cartilage were non-significantlydecreased by 19% and 69%, respectively. All sections had cartilagehypertrophy. Central MFC thickness in these sections was significantlyincreased by 106%.

Conclusions:

Abnormal clinical signs were mild and consistent with those typicallyseen in rabbits following knee surgery and/or as a result of repetitivesample collection, either blood collected from the lateral ear veins orsynovial fluid collected from the knee joint. Comparison ofintercondylar groove healing of animals in Group 1 and Group 2 showedgood spontaneous filling of the groove with what grossly appears to becartilage. There is a suggestion that microfracture may enhancecartilage regrowth in the groove since 9/10 animals in Group 2 hadfilling of the groove with cartilage compared to 7/10 animals in Group1, but this difference is slight.

Likewise, there is no difference between ICRS or osteophyte scoresbetween the two groups so the quality of cartilage healing was good, andthere was little stimulation of osteophyte formation in either group. Ofthe remaining groups treated with rhFGF-18, it appears that Group 4(intercondylar groove and three monthly intra-articular injections of100 μg of rhFGF-18) showed the best response, with 6/9 animals havingthe intercondylar groove filled with cartilage that appeared nearlynormal to slightly roughened or granular in gross appearance. Inaddition, Group 4 had the next to lowest ICRS cartilage score(2.1+/−0.3) of the four groups treated with rhFGF-18, and this group hadthe lowest osteophyte score (3.8+/−0.8) of any of the rhFGF-18 groups.In contrast, Group 6 (intercondylar groove with microfracture and threemonthly intra-articular injections of 100 μg of rhFGF-18) hadintercondylar groove filling in only 3/10 animals, the ICRS score wasthe highest of any group (2.4+/−0.2) and the osteophyte score was thesecond highest (5.1+/−0.7) of any rhFGF-18 treated group. Likewise,Group 5 (intercondylar groove with microfracture and three weeklyintra-articular injections of 100 μg of rhFGF-18) had the highestosteophyte score of any group (5.7+/−0.7).

Beneficial effects were greater when treatment was given over anextended time period (3 months vs. 3 weeks), and effects were moreevident in rabbits with microfractures than in rabbits withoutmicrofractures.

Example 4

Method:

Porcine chondrocytes were isolated from the cartilage of a femoral headof a pig hip (pigs were approximately one year old). After dissection ofthe joints, the cartilage was harvested and digested for 45 minutes withcollagenase 0.25% ( 1/10 dilution of collagenase NBG4 2.5% in HAM's F12)at room temperature. The loosened cells were discarded and the cartilagefurther digested overnight with collagenase 0.1% ( 1/25 dilution ofcollagenase NBG4 2.5% in HAM's F12) at 37° C. to extract thechondrocytes. For this study, the chondrocytes were cultured in amonolayer.

Primary articular porcine chondrocytes were stimulated or not with 10ng/mL IL1α and were either treated immediately or 6 hours later withanakinra 100 ng/mL and/or trFGF-18 100 ng/mL. Each condition wasperformed in 4 or 6-plicate. As a control, cells were also cultured withanakinra alone, trFGF-18 alone or in the absence of any treatment(medium).

Pre-culture: After cell isolation porcine chondrocytes were inoculatedat 20,000 cells/cm² and cultivated for one week in complete HAM's F12.Cells were then harvested with accutase, counted and used as describedbelow.

For cells treated directly with anakinra: Chondrocytes were inoculatedin a 24-well plate at 15,000 cells/well in 0.5 mL of complete HAM's F12.Then 0.25 mL of trFGF-18 400 ng/mL and/or 0.25 mL of anakinra 400 ng/mLwere added to the cells. In the challenged samples, 10 μL of IL1α at1,000 ng/mL were also added to the cells. trFGF-18, anakinra and IL1αwere all diluted in complete HAM's F12. The final volume in the culturewell was adjusted to 1 mL with complete HAM's F12. Final concentrationswere 10 ng/mL IL1α, 100 ng/mL trFGF-18 and 100 ng/mL anakinra and thetotal culture time was eight days. A complete medium change wasperformed after four days. N=4.

For cells treated with anakinra and trFGF-18 6 hours after the IL1αchallenge: Chondrocytes were inoculated in a 24-well plate at 15,000cells/well in 1 mL of complete HAM's F12 containing or not containingIL1α 10 ng/mL. After 6 hours the medium was removed and replaced with0.25 mL trFGF-18 400 ng/mL and/or 0.25 mL of anakinra 400 ng/mL. In thechallenged samples, 0.25 mL of IL1α at 40 ng/mL was also added to thecells. trFGF-18, anakinra and IL1α were all diluted in complete HAM'sF12. The final volume in the culture well was adjusted to 1 mL withcomplete HAM's F12. Final concentrations were 10 ng/mL IL1α, 100 ng/mLtrFGF-18 and 100 ng/mL anakinra and the total culture time was eightdays. A complete medium change was performed after four days. N=6.

After eight days of culture, cells were harvested with accutase andresulting cell suspensions were analyzed for cell concentration and cellviability with a ViCell™ cell analyzer (from Beckman Coulter).Statistical analysis consisted of a 1-way ANOVA followed by a Dunnettpost hoc analysis.

Results and Conclusions:

As expected, rhFGF-18 was found to increase porcine chondrocyteproliferation in the absence of IL1α. In both experiments in comparisonwith the control (medium, no IL1α) an 11.25 and 7.75-fold increase inthe cell number was observed in the presence of rhFGF-18 100 ng/mL after8 days of culture (FIG. 6). However, in the presence of IL1α 10 ng/mL,the stimulation of the proliferation by rhFGF-18 was only 2.38 and2.44-fold, respectively, in comparison to the control (medium, IL1α, 10ng/mL) for both experiments. Anakinra 100 ng/mL, for both the direct andthe delayed treatments, was shown to fully restore rhFGF-18 activity.The cell density for cells cultured with rhFGF-18 and without IL1α orchallenged with IL1α but treated with anakinra was found not to bestatistically different. Finally, anakinra alone had no effect onchondrocyte proliferation.

In the present study, anakinra 100 ng/mL has been shown to fully blockthe inhibitory effect of IL1α on the proliferative activity of rhFGF-18.This in accordance with the fact that anakinra is an IL1 receptorantagonist, blocking the inflammatory signaling triggered by IL1. It hasbeen presently demonstrated that anakinra restores the proliferativeeffect of rhFGF-18 in porcine chondrocytes challenged with IL1α.

Example 5

Method:

Male Lewis rats underwent surgery to induce a medial meniscal tear inthe right knee joint. Animals were dosed i.a. with FGF-18 (10 μg/joint)on days 21, 28, and 35 and dosed orally with diclofenac (either 1 mg/kgor 3 mg/kg) or vehicle on days 21-23, 28-30, and 35-37, then terminatedon day 42 or day 63. Serum was collected and evaluated for alpha 2macroglobulin (α2MG) levels on days −3, 21, 42, and 63. α2MG is aninflammation serum biomarker. The link between α2MG and inflammation hasalready been shown (Kuribayashi et al., 2013).

Results

FGF-18+vehicle controls had mild to moderate swelling on day 24, whichincreased to severe by day 36, although no limping was observed. Kneeswelling diminished slightly to marked severity beginning on day 44.Caliper measurements supported these observations, with significantdifferences between right and left knees at day 21 and later timepoints, but not at the baseline. Animals given 3 mg/kg of diclofenac hadsignificantly reduced swelling scores on days 24-29, 31, and 36-43,compared to the vehicle controls. Knee caliper measurements weresignificantly reduced on day 42, but were not significantly affected atany other time point. Animals given 1 mg/kg of diclofenac hadsignificantly reduced swelling scores on days 24-29, 31, and 36-43,compared to the vehicle controls. Knee caliper measurements weresignificantly reduced on day 42, but were not significantly affected atany other time point (data not shown).

α2MG levels were higher on day −3 (pre-surgery) than at day 21post-surgery. Levels in animals given 3 mg/kg of diclofenac wereslightly but significantly reduced compared to those of the vehiclecontrols at the pre-surgery time point. Levels on day 21, prior totreatment, were essentially the same for all groups. On day 42, thedisease controls had markedly elevated α2MG levels (approximately fourtimes the day 21 levels), which was a significant difference from thetwo diclofenac groups. On day 63, levels for all groups were similaragain (FIG. 7).

Conclusions:

Overall, results of this study indicate that administration of 1 or 3mg/kg of diclofenac in rats with medial meniscal tears that were alsogiven FGF-18 significantly reduced knee swelling, based on both clinicalobservations and caliper measurements. These effects were evident forapproximately one week after the final dose of diclofenac. Serum levelsof α2MG tracked with the vehicle controls on days −3, 21, and 63;however, a sharp reduction was seen on day 42 as a result of a massivespike in the control levels.

Example 6

Method:

The goal of this study was to evaluate the effect of differentintracycle dosing frequency of the same total dosage (3×1 μg) ofsprifermin on cartilage volume in a rat model of OA. The anteriorcruciate ligament transection (ACLT) with resection of the medialmeniscus (tMx) was performed as disclosed in Example 1. Joints from maleLister Hooded Rats (200-260 g, Charles River) were used in this study.Animals were euthanized 18 weeks post-surgery.

The changes in the joint are related to clinical assessments in OA(cartilage loss, osteophytes, subchondral sclerosis). One μg spriferminin saline was injected three times either weekly, every 2nd, every 3rd,every 4th or every 5th week and compared to appropriate vehicle groups.Body weight, joint diameters and clinical health scores wereinvestigated weekly.

Results

Results shown that intra-articular injections had no influence on thebody weight gain. No significant drug or treatment regime effect wasseen on the in-life parameter. However, groups injected every secondweek with saline (n=4) had the highest body weight gain tendency (datanot shown).

With injections in week 3, 4 and 5, joint diameters were significantlyhigher than after injections in week 3, 8 and 13 (see FIG. 8). Nosignificant differences were seen between injections every 2nd, 3rd or4th week. When injected only every 5th week AUC of joint diameters wasthe same as with saline injections. So, in tendency, the joint diameterAUC decreases with lower injection frequency.

The quantitative histological analysis via stereology reviled severeOA-like changes in the affected joints. rhFGF-18 prevented more or lesscomplete cartilage denudation. One μg/joint and injection given onceevery 3rd week (three injections in total) resulted in pharmacologicallymeaningful and statistically significant difference over saline(endpoint: cartilage volume on medial tibia, see FIG. 9).

Conclusions:

Lower injection frequencies than once weekly over three weeks seem toallow better recovery from joint swelling after injection. All groupstreated with rhFGF-18 displayed structural benefits illustrated byhigher cartilage volume values compared to placebo. However, under thecircumstances of a time-fixed study (same study duration for allanimals) and a fixed dosis, the injection frequency once every 3rd weekresulted in the most beneficial structural outcome. It can be speculatedthat longer pauses between injections of rhFGF-18 are more beneficialbecause of better translation of chondrocyte proliferation intoextracellular matrix production compared to the once weekly over threeweeks injection regimen.

The resulting bell-shaped curve is a common observation for efficacyreadouts of growth factors.

REFERENCES

-   1. Ellsworth et al., 2002, Osteoarthritis and Cartilage, 10: 308-320-   2. Shimoaka et al., 2002, JBC, 277(9):7493-7500-   3. WO2008/023063-   4. WO2004/032849-   5. WO98/16644-   6. WO2006/063362-   7. Custers et al., 2007, Osteoarthritis and Cartilage, 15:1241-1248-   8. Lotz, 2010, Arthritis Research Therapy, 12:211-   9. Moore et al., 2005, Osteoarthritis and Cartilage, 13:623-631-   10. Arnaud-Dabernat et al., 2008, Journal of Cellular Physiology,    216:261-268-   11. The Merck Manual, 17^(th) Edition, 1999-   12. Getgood et al., 2010, P116, ICRS Meeting 2010, Barcelona-   13. ICRS publication: see Worldwide Website:    cartilage.org/_files/contentmanagement/ICRS_evaluation.pdf, page 13-   14. Bingham, 2002, J. Rheumatol., 29:3-9-   15. Lee et al., 2013, Gene, 527:440-447-   16. Bresnihan, 2002, Ann. Rheum., 61:ii74-ii77-   17. St. Clair, 2002, J. Rheumatol., 29:22-26-   18. Mertens et al., 2009, J. Rheumatol., 36(6):1118-1125-   19. Chevalier et al., 2009, Arthritis & Rheumatism, 61(3):344-352-   20. Kuribayashi et al., 2013, Inflammation, 36(6):1448-52

The invention claimed is:
 1. A method for the treatment of a cartilagedisorder comprising administering to a subject having a cartilagedisorder an FGF-18 compound selected from the group consisting of: apolypeptide comprising residues 28-207 of SEQ ID NO:1; and a polypeptidecomprising SEQ ID NO:2, wherein said FGF-18 compound is administered atleast twice per treatment cycle, said administrations being separated byabout at least 3, 4 or 5 weeks.
 2. The method according to claim 1,wherein said administrations are separated by about 3 weeks or by about4 weeks.
 3. The method according to claim 1, wherein saidadministrations are separated by about 1 month.
 4. The method accordingto claim 1, wherein the FGF-18 compound is administered at least 3 timesor at least 4 times per treatment cycle.
 5. The method according toclaim 1, wherein treatment cycles are repeated after 2, 3, 4, 5 or 6months.
 6. The method according to claim 1, which comprises 1, 2 or 3treatment cycles per year.
 7. The method according to claim 1, whereinthe FGF-18 compound is administered intra-articularly.
 8. The methodaccording to claim 1, wherein the FGF-18 compound is administered at adose of 3-300 mcg per single intra-articular administration.
 9. Themethod according to claim 8, wherein the FGF-18 compound is to beadministered at a dose selected from about 3, 10, 20, 30, 40, 50, 60,90, 100, 120, 150, 180, 200, 240 or 300 mcg per single intra-articularadministration of the FGF-18 compound.
 10. The method according to claim1, wherein the cartilage is articular cartilage.
 11. The methodaccording to claim 1, wherein the cartilage disorder is osteoarthritis.12. The method according to claim 1, wherein the cartilage disorder iscartilage injury.
 13. The method according to claim 1, wherein theFGF-18 compound is a polypeptide comprising residues 28-207 of SEQ IDNO:1.
 14. The method according to claim 1, wherein the FGF-18 compoundis administered together with an anti-inflammatory drug.
 15. The methodaccording to claim 14, wherein the anti-inflammatory drug is anakinra ordiclofenac.
 16. The method according to claim 14, wherein theanti-inflammatory drug is administered at a dose of 0.001-500 mg persingle administration.
 17. The method according to claim 16, wherein theanti-inflammatory drug is administered at a dose selected from 0.03,0.1, 0.25, 0.3, 0.5, 1, 1.5, 2, 5, 10, 15, 20, 30, 40, 50, 60, 70, 80,90, 100, 150, 200, 250 or 300 mg per single administration.
 18. Themethod according to claim 1, wherein the FGF-18 compound is administered3 times per treatment cycle, said administrations being separated byabout 3 weeks.
 19. The method according to claim 1, wherein the FGF-18compound is administered 3 times per treatment cycle, saidadministrations being separated by about 4 weeks.
 20. The methodaccording to claim 1, wherein the FGF-18 compound is administered oncemonthly for 3 consecutive months.
 21. The method according to claim 1,wherein the FGF-18 compound is a polypeptide comprising SEQ ID NO:2.